Murine DNA polymerase.alpha-primase initiates RNA-primed DNA synthesis preferentially upstream of a 3'-CC(C/A)-5' motif.
We find that the purified murine DNA polymerase.alpha-primase complex exhibits the greatest affinity for DNA templates in which CCC occurs 10 nucleotides downstream of a DNA primase initiation site (Km = 6.6 +/- 0.3 pM). Templates with 3'-CCA-5' at this position are less efficiently utilized (Km = 16 +/- 4 pM). Point mutations that disrupt the 3'-CC(C/A)-5' motif further decrease the affinity for DNA approximately 7-fold (Km = 105 +/- 58 pM). Mutations at the primase start site reduce Vmax 2-fold. Template pyrimidines are required for priming, and initiation with ATP is preferred to initiation with GTP. We conclude that a component of the DNA polymerase.alpha-primase complex recognizes a 3'-CC(C/A)-5' motif in the DNA template, downstream of a primase start site, and that this interaction controls site selection and frequency of initiation by DNA primase.
Davey
S K
SK
Department of Biochemistry, University of Western Ontario, London, Canada.
Faust
E A
EA
D001483:Base Sequence; D003001:Cloning, Molecular; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D008915:Minute Virus of Mice / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D015183:Restriction Mapping; D013698:Templates, Genetic
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases